Peptides can be easily denatured due to their low stability, lose their activities via degradation by in-vivo proteolytic enzymes, and are so small so as to be easily removed through the kidney. Accordingly, in order to maintain the blood levels and the titers of a pharmaceutical drug containing a peptide as a pharmaceutically active component, it is necessary to frequently administer the peptide drug to a patient.
However, most peptide drugs are administered as injection preparations and frequent administration is necessary for the maintenance of the blood level of the physiologically active peptides, thus causing a severe pain for the patients. To solve these problems, many efforts have been made to increase the stability of peptide drugs in blood and to maintain the drugs in the blood at high level for a prolonged period of time, thereby maximizing the pharmaceutical efficacy of the drugs.
In particular, the long-acting preparations of such peptide drugs therefore need to increase their stability while simultaneously maintaining their titers at sufficiently high levels without causing any immune responses in patients. In this regard, as methods for stabilizing the peptides, and inhibiting the degradation by a proteolytic enzyme, attempts have been made to modify specific amino acid sequences which are sensitive to proteolytic enzymes.
For example, GLP-1 (7-37 or 7-36 amide), which has a therapeutic effect of lowering the blood glucose level for treating type 2 diabetes, has a short physiological half-life of about 4 minutes or less, due to loss of the titers of GLP-1 through the cleavage between the 8th amino acid (Ala) and the 9th amino acid (Asp) by a dipeptidyl peptidase IV (DPP IV). In this regard, GLP-1 derivatives, in which Ala8 is substituted with Gly, Leu, or D-Ala to increase the resistance to DPP IV while maintaining the physiological activity, have been developed. Moreover, the N-terminal amino acid of GLP-1, His7, is critical for the GLP-1 activity, and serves as a target for DPP IV. Accordingly, the N-terminus is modified to an alkyl or acyl group, and His7 is subjected to N-methylation or alpha-methylation, to increase its resistance to DPP IV, and to maintain its physiological activity. Although it was confirmed that the stability was improved due to an increase in their resistance to DPP IV there was also a report that the receptor affinity of the derivatives obtained by modifying His7 was considerably reduced and the secretory capacity of cAMP was reduced at the same concentration (see, Galiwitz al., Regulatory Peptide 79: 9-102, 1999; Galiwitz et. al., Regulatory Peptide 86: 103-111, 2000).
In addition, among the insulinotropic peptides such as GLP-1, exendin-4 is composed of a sequence of His-Gly instead of His-Ala, which is a sequence of GLP-1 acting as a substrate for DDP IV. Accordingly, exendin-4 has resistance to DPP IV and higher physiological activity than that of GLP-1. Thus, exendin-4 has a longer in-vivo half-life than that of GLP-1. However, although the in-vivo half-life of exendin-4 is longer than that of GLP-1, commercially available exendin-4 (exenatide) must be administered to a patient twice a day via injection, and this is a major inconvenience for patients.
Under these circumstances, the present inventors have made various efforts to increase the activities and stability in blood of insulinotropic peptides and discovered that insulinotropic peptide derivatives with a modified N-terminal charge can exhibit superior pharmacokinetics and higher insulinotropic activities compared to those of a native insulinotropic peptide, thereby completing the present invention.